Mosquito borne diseases include malaria, yellow fever, and dengue fever. Malaria occurs in over 100 countries and approximately 40% of the world's population is at risk. According to the CDC, more than 1 million deaths each year are caused by malaria, mostly small children in African areas south of the Sahara. Ticks can transmit encephalitis, Lyme disease and Typhus fever. Filariasis can be transmitted by blackflies, leishmaniasis by sandflies, sleeping sickness by Tsetse flies, and Chagas disease by assassin bugs. Lice can spread typhus fever and fleas are the well-known vector for plague. While the common house fly (Musca domestica) does not bite, it can transmit typhoid fever, cholera, dysentery, pinworms, hookworms, and certain tape worms. Stable flies (biting house flies) can also transmit filth-related disease. Reducing bites or reducing fly landings reduces the chances of getting these diseases, and insect repellents (e.g. DEET or N,N-diethyl-m-toluamide) are recommended for this purpose.
Insecticides (e.g. pyrethrum or permethrin) used as sprays or bed-net impregnants also reduce bites. DEET is effective in repelling mosquitoes, but it is a poor fly repellent. Its efficacy against mosquitoes is limited to a few hours duration by high evaporative loss from the skin surface (about 70% of applied dose) and also by significant absorption into the skin (approximately 20%). The usefulness of permethrin for insect control rests largely on its ability to cause incapacitation and death after insects make contact with treated surfaces or sprays; its low vapor pressure (about 10−8 mm Hg@20° C.) makes it relatively useless as a repellent. Permethrin's insecticidal properties are maximized in emulsion formulations; however, these formulations also maximize skin absorption (about 30%) and human skin contact should be avoided. Permethrin's efficacy has been reduced by insect resistance (Saaverdra-Rodriguez, K., C. Strode, A. F. Suarez, I. F. Salas, H. Ranson, J. Hemingway and W C. Black IV. Quantitative Trait Loci Mapping of Genome Regions Controlling permethrin resistance in the mosquito Aedes aegypti. Genetics, 180: 1137-1152, 2008).
Blood-sucking insects use a variety of physical and chemical cues to seek out a host. Their behavior can also be modified by emissions or pheromones from themselves or other species. A better understanding of the chemical-mediated behavior has lead to the development of alternate control measures, such as mating disruption.
A variety of factors and chemicals, including fatty acids, have been implicated or suggested as mosquito host attractants, and the literature contains many contradictory reports. Brown lists a number of factors involved in the attraction of mosquito to humans (in order of importance): moisture, convective heat, carbon dioxide, movement, contour or increase in black-white interfaces, and reflectivity (Brown A. W. A., H. P. Roessler, E. Y. Lipsitz and A. G. Carmichael. Factors in the attractiveness of bodies for mosquitoes. The Canadian Entomologist 96:102-103, 1964).
Since 1995, we have been investigating fatty acids as repellants or biopesticides against insects and arthropods of medical or economical importance and acknowledge past support from the USDA (Reifenrath, W G. Natural Fly Repellent for Livestock. SBIR Phase II Final report, CSREES Award No. 2003-33610-13044, Feb. 1, 2007), US Army (Reifenrath, W G. Development of an Insect Repellent Based on Human Skin Emanations. Final Report, DAMD17-96-C-6046, October, 1996), and DoD (Reifenrath, W G. New Repellent Combination against Flies and Mosquitoes. Final Report, USAMRMC Award No. W81XWH-04-1-0787, Final Report, December 2006). Our approach has been utilitarian; that is, to first focus on low-cost formulations that had a reasonable chance of regulatory approval and to demonstrate that these formulations were effective in laboratory (Mullens, B A, Reifenrath, W G, and Butler, S M. Laboratory repellency trials of fatty acids against house flies, horn flies, and stable flies (Diptera: Muscidae). Pest Management Science, 65: 1360-1366, 2009; Reifenrath, W G. Natural Insect and Arthropod Repellent. U.S. Pat. No. 6,306,415 B1, Oct. 23, 2001) and field studies for insect control (Chansang, U. and Mulla, M. S. Field Evaluation of repellents and insecticidal aerosol compositions for repelling and control of Siphunculina funicola (Diptera: Chloropidae) on aggregation sites in Thailand. J. Am. Mosq. Control Assn. 24: 299-307, 2008; Reifenrath, W G. Natural Fly Repellent for Livestock, Stratacor, Inc. http://www.reeis.usda.gov/web/crisprojectpages/200581.html). Stratacor has an application approved by the U.S. EPA for a formulation of three medium chain fatty acids (octanoic, nonanoic, decanoic acids, trademarked C8910) for fly and lice control on cattle, as well as an approval for use of the three fatty acids on food commodity animals (Anonymous. Application to Register C8910 Fly Repellent Oil, Stratacor, Inc., Richmond Calif., Jan. 18, 2008; Anonymous. Notice of Pesticide Registration, C8910 Fly Repellent Oil, EPA Reg. No. 84893-1, Biopesticides and Pollution Prevention Division, Office of Pesticide Programs, U.S. Environmental Protection Agency, Washington, D.C., Oct. 13, 2009; Anonymous. Notice of Filing of a Pesticide Petition for Residues of Pesticide Chemicals in or on Various Commodities. US Environmental Protection Agency, Federal Register, Vol. 73, No. 54, Wednesday, Mar. 19, 2008; Anonymous. Memorandum, Tolerance Exemption Petition for the Active Ingredients C8-C10 n-carboxylic acids (octanoic acid, nonanoic acid, and decanoic acid), Biochemical Pesticides Branch, Biopesticides and Pollution Prevention Division, US Environmental Protection Agency, Washington, D.C., Oct. 8, 2009). These fatty acids have been approved by the US FDA as food additives (flavors) in the U.S. since 1965 and are categorized as “Generally Recognized as Safe”. C8910 can be formulated for direct use on the skin, and a topical formulation is being advanced for human use in Africa. However, user compliance with topical repellents, even in the U.S. military, is low (about 30%). Therefore, our long term goal is to advance safe and cost-effective C8910 formulations for both direct skin application and area treatments or space sprays to control the spread of malaria by the Anopheles mosquito, filth related disease by house and stable flies, and tick borne diseases. The fatty acids comprising C8910 are inexpensive commodity chemicals that come from palm kernel oil or coconut oil (as a byproduct of coconut production) and from cattle tallow. These compounds could eventually be produced in developing countries.
Fatty acids of chain length 8 (caprylic or octanoic) to 11 (undecanylic acid) were reported to be toxic to house fly larvae (Quraishi, M. S. and A. J. Thorsteinson. Toxicity of some straight chain saturated fatty acids to house fly larvae. J. Econ. Entomol. 58: 400-402, 1965). The fatty acids octanoic, nonanoic, and undecylenic were found to have ovicidal activity against eggs of Aedes aegypti; the author suggested that the fatty acids exerted a “smothering” effect or interference with respiration (Cline, R. E. Lethal effects of aqueous formulations containing fatty amines or acids against eggs and larvae of Aedes aegypti. J. Econ. Entomol. 65: 177-181, 1972). The straight chain fatty acids (C7 to C11, but not C12) were found to be toxic to Aedes aegypti instar IV larvae and pupae (Quraishi, M. S. and A. J. Thorsteinson. Effect of synthetic queen substance and some related chemicals on immature stages of Aedes aegypti. J. Econ. Entomol. 58: 185-187, 1965). Caproic (C6) through capric acid (C10) were found to have optimal larvicidal activity for housefly larvae, with declining activity for undecanoic acid (C11) and lauric acid (C12) (Levinson, Z. H. and K. R. Simon Ascher. Chemicals affecting the preimaginal stages of the housefly. Rivista DI Parassitologia. 15: 111-119, 1954).
We have shown that C8910 is an effective mosquito repellent for use on the skin, and can also repel ticks, biting flies and significantly reduce the number of fleas and lice on animals. As a vapor phase repellent against mosquitoes, a number of straight and branched chain fatty acids have activity in olfactometer tests with Aedes aegypti mosquitoes (FIG. 1), with C8, C9, and C10 sitting at or near the top of the repellency “dome”. As frequently seen in homologous series of insect repellents (Skinner, W. A. and Johnson, H. L. The design of insect repellents. In: Drug Design, Vol. 10, E J Ariens, Ed., Academic Press, New York, pp. 277-305, 1980), activity at lower chain length is limited by lack of persistence due to high volatility. Activity at higher chain length is limited by lack of volatility, as mosquito repellents are required to have a minimum effective evaporation rate (Reifenrath, W. G. and Robinson, P. B. In Vitro Skin Evaporation and Penetration Characteristics of mosquito repellents. J. Pharm. Sci, 71:1014-1018, 1982; Reifenrath, W. G. and Spencer, T. S. Evaporation and penetration from skin. In: Percutaneous Absorption Mechanisms Methodology Drug Delivery, R L Bronaugh and H I Maibach, Eds., 1st Ed., Marcel Dekker, New York, pp. 305-325, 1985).
The fatty acids comprising C8910 can penetrate the skin after topical application and can also “back diffuse” or reach the skin surface after application to the visceral side of the excised skin (Reifenrath, W. G. Unpublished data, Stratacor, Inc., December, 2005-February, 2006). However, these compounds are not effective insect repellents after oral administration to cattle (Personal Communication, David Boxler, Dept. of Entomology, University of Nebraska West Central Research and Extension Center, North Platte, Neb., Jun. 27, 2006), as they are metabolized too quickly to be excreted via the skin (Van Den Driessche, M., K. Peeters, P. Marien, Y. Ghoos, H. Devlieger, and G. Veerman-Wauters. Gastric emptying in formula-fed and breast-fed infants measured with the 13C-octanoic acid breath test. J. Pediatr. Gastroenterol. Nutr. 29: 46-51, 1999).
While conducting field studies on livestock, we were surprised to find that dusting cattle with C8910 offered protection (about 90% reduction in total body fly counts) against biting (horn) flies equal to the organophosphorus insecticides coumaphos (Co-ralR) and tetrachlorvinphos (RabonR) (FIG. 2). These results have been reproduced by different investigators at different locations where temperature and humidity are high. In this type of test, C8910 was formulated as a dust and cattle self-treated by walking under a dust bag en-route to water. If C8910 were acting just as a repellent, the flies would simply have moved from the treated area (back) to the untreated areas (belly and legs), resulting in no net reduction in fly count. A direct toxic effect of C8910 on the flies could account for the efficacy, but this was not obvious at the time of the test. This prompted a laboratory examination of the incapacitative and toxic effects of C8910 on Aedes aegypti mosquitoes, which showed that direct contact (aqueous suspension of C8910 wettable powder on treated filter paper in Petri dishes) caused incapacitation in 10 minutes at a dose of 4.7 ug/cm2, in 25 minutes at a dose of 2.35 ug/cm2, and while a still lower dose of 1.18 ug/cm2 did not cause incapacitation, it stopped spontaneous movement of mosquitoes, with negative consequences for mating and reproduction. Qualitatively similar activity was found in laboratory studies with house flies, stable flies, and horn flies (FIGS. 3-10), with house flies being least sensitive to the incapacitative and toxic effects of C8910. An initial report indicated that the Tsetse fly was not repelled by C8910 dust formulations (Personal Communication, Serap Aksoy, Dept. of Epidemology and Public Health, Yale School of Medicine, New Haven, Conn., Apr. 21, 2003), but a later report showed that a 15% mineral oil and an 0.3% wettable powder dispersion were effective in incapacitating or killing the Tsetse fly (Personal Communication, Brian Weiss, Dept. of Epidemology and Public Health, Yale School of Medicine, New Haven, Conn., Aug. 3, 2009). Tsetse fly repellency may have been compromised by incapacitation, an effect that was not appreciated until recently. In tests conducted in free choice cages as described by Mullens et al. (Mullens, B A, Reifenrath, W G, and Butler, S M. Laboratory repellency trials of fatty acids against house flies, horn flies, and stable flies (Diptera: Muscidae). Pest Management Science, 65: 1360-1366, 2009), the German cockroach became incapacitated and then died after roaming into floor areas treated with 15% C8910 in attapulgite clay (FIG. 11).
We have found that incapacitative and toxic effects of fatty acids on mosquitoes are highly dependent on the type of fatty acid and the formulation. When moisture is introduced in the form of aqueous dispersions of powder formulations, house fly and mosquito incapacitation and death result. Likewise, with mineral oil formulations. Both of these formulations enhance the mammalian skin absorption of C8910 and likely enhance fly uptake and absorption. Formulations made from emulsifiable concentrates (EC) promote skin absorption and enhance fly toxicity of permethrin (Reifenrath, W G. Enhanced skin absorption and fly toxicity of permethrin in emulsion formulation. Bull. Environ. Contam. Toxicol. 78: 299-303, 2007). Likewise, formulations of C8910 in EC cause house fly and mosquito toxicity. The well-known insecticidal properties of soaps (sodium or potassium salts of fatty acids), where the fatty acid is ionized at basic pH, is probably due to a different mechanism related to surfactant effects. Indeed, the ionic and non-ionic surfactants contained in an emulsifiable concentrate vehicle for permethrin (Evercide emulsifiable concentrate without permethrin, MGK Corp, Minneapolis) had no fly incapacitative effects. Incapacitation was not simply due to acidic pH, as lactic acid, a low molecular weight carboxylic acid, was nontoxic to flies in these assays. Lauric acid, with increased carboxylic acid carbon chain length beyond those comprising C8910, was also non-toxic in EC formulation. The insect repellent DEET did cause fly and mosquito mortality after 24 hours at 47 ug/cm2 (the fatty acid dose that caused 100% immediate fly and mosquito incapacitation and 80-100% mortality at 24 hours), but by a mechanism that did not involve immediate incapacitation. As a test of a sodium ion channel blocker, freshly prepared aqueous emulsions of lidocaine free base (47 ug/cm2) or 15% concentration in mineral oil did not cause fly incapacitation in these assays. At a dose of 4.7 ug/cm2, permethrin caused irreversible fly and mosquito incapacitation, while the same low dose of C8910 showed reversible incapacitation against the stable fly and horn fly (FIGS. 9,10).
Observationally, the fatty acids comprising C8910 appear to have a selective repellent and/or toxic effect against mosquitoes and a variety of other flies, as spiders, bumble bees, honey bees, and wasps are unaffected. Ticks (Ixodes pacificus or Dermacentor) placed in the barren center of free choice cages and allowed to roam into quadrants of C8910 laden granules or carrier-only treated quadrants typically get within ½ inch of the repellent granules, do an about-face, and proceed into and through control areas and climb the walls of the cage, when they were captured by the investigator and placed back into the starting center. Occasionally, a tick will wander between repellent granules, and no longer having a choice between repellent and non-repellent areas, may continue to wander aimlessly in contact with the repellent, until it stopped all movement and eventually died. Forced exposure of ticks (Ixodes pacificus) to a 0.3% formulation of C8910 in Kaolin-P water dispersion for 10 minutes in a Petri dish resulted in 50% incapacitation, 100% incapacitation in 40 minutes and 100% mortality in 24 hours. No incapacitation or toxicity was observed with control (kaolin clay water dispersion) over 24 hours. A similar type of behavior was observed with the German cockroach, as noted above (FIG. 11). While these are descriptive accounts, they go to the heart of the proposed mechanism of fatty acid toxicity to susceptible insects—an indirect effect based on interference with the insect's ability to navigate, giving the appearance of death before actual death (the “living dead” or paralysis syndrome). At low levels comparable to natural pheromone concentrations, the fatty acids of C8910 may actually be attractive, and this has been observed with ants. Higher air concentrations of subject fatty acids may overload sensory mechanisms, and insects are repelled. If C8910 is forced into contact with susceptible insects in a spray formulation that promotes adherence to the insect or absorption by the insect (in effect surrounding the insect with C8910), reversible incapacitation may result at lower doses to the insect. At higher doses, the incapacitation becomes irreversible and the insect eventually dies.